Method for determining the total iron-binding capacity of blood serum

ABSTRACT

The use of an aqueous solution of a polycarboxylic acid as the means of initially releasing the bound iron from the transferrin in a blood serum specimen has been found advantageous in determining the total iron-binding capacity (TIBC) of blood serum. Reliable and reasonably accurate results are obtained even where the polycarboxylic acid reagent has been stored for an extended period prior to use in the test method.

United States Patent Ogawa et al.

METHOD FOR DETERMINING THE TOTAL IRON-BINDING CAPACITY OF BLOOD SERUM Inventors: Hiroshi Ogawa, Kashiwa; Kazuo Nitta, Yachiyo; Nobuhiko Nakazawa, Urawa; Kentaro Maki, Tokyo, all of Japan Assignee: Mallinckrodt, Inc., St. Louis, Mo.

Filed: Feb. 11, 1974 Appl. No: 441,165

Related [1.8. Application Data Continuationin-part of Ser, No. 371,05l, June 18, I973. abandoned Foreign Application Priority Data Marl 5, I973 Japan 4825926 US. Cl 23/230 B; 250/303, 424/1 int. Cl Goln 23/00; GOln 33/16 Field of Search 23/230 B; 424/l Dec. 9, 1975 [56] References Cited UNITED STATES PATENTS 3,537,822 l1/l970 OMalley i. 23/230 B 3,709,985 l/l973 Burke 3,773,467 ll/l973 Yang et al 23/230 B Primary ExaminerMorris O. Wolk Assistant Examiner-Sidney Marantz Attorney, Agent, or Firm-Keenig, Senniger, Powers and Leavitt [57] ABSTRACT 6 Claims, N0 Drawings METHOD FOR DETERMINING THE TOTAL IRON-BINDING CAPACITY OF BLOOD SERUM CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of our application Ser. No, 371,051, filed June 18, 1973, now abandoned.

BACKGROUND OF THE INVENTION The present invention relates to a method for determining the total iron-binding capacity (referred to as TIBC hereinafter) of serum protein in blood.

In the living body, particularly in the blood serum, there exists a specific serum protein called transferrin and it has a specific iron-binding ability and the determination of the total capacity is very important in the clinical diagnostic area. The normal transferrin is partially saturated by iron ions and the quantity of iron is known as serum iron (SI). Moreover, transferrin can be saturated by additional iron ions. The corresponding additional capacity is called unsaturated iron-binding capacity (referred to as UIBC hereinafter). Therefore, TIBC is equal to the total amount of SI and UIBC.

Up to this time, several methods have been known for determining TIBC. Out of these methods, the Burke method (US. Pat. No. 3,709,985) and the Saito method (Nippon Ketsueki Gakukai Zasshi Vol. 33, pages 555-559 (l970)and Journal of Nuclear Medicine Vol. 13, pages 489-492 (1971) are closely related to this invention. 7 A

The Burke method comprises the following steps. 'llhe bound iron (SI) is released by addition of hydrochloric acid to serum and the released iron is removed with calcium carbonate adsorbent. An excess of radioactive iron, and then, an ion-exchange resin are added to the serum. The mixture is incubated and the initial radioactivity of the mixture is measured. After the measurement, the resin is taken out of the mixture, washed and the radioactivity of the resin is measured.

The Saito method comprises the following steps. Serum is treated with an ascorbic acid solution to release iron. The released iron is removed from the serum with ion-exchange resin. Radioactive iron ions are added to the serum and the initial radioactivity of the mixture is measured. Unbound iron ions are removed from the mixture with ion-exchange resin and the residual radioactivity of the serum is measured. This method is a very excellent and convenient one. However, when the measurement is carried out using a previously prepared reagent of ascorbic acid (for example, several weeks before the measurement), the TIBC value shows considerably higher than the value measured according to other classical methods (for example, colorimetry with bathophenanthroline: W. N. M. Ramsey, Clin. Chim. Acta Vol. 2 pages 221-226 i957) and the exact TIBC value could not be realized (see Example I). In practicing the measurement of TIBC in hospitals, it is desirable to use previously prepared reagents from the viewpoint of treating radioactive substances and having to carry out the measurement rapidly and punctually. The present inventors have studied Saitos method to find the reason why the higher value results and have concluded that it is caused by instability of aqueous ascorbic acid solution which had been previously prepared and used in the first step in the Saito method. Accordingly, the previous preparation of ascorbic acid causesran inclination toward the higher TIBC value for measurement.

Thereupon, the inventors have found a desirable substitute for ascorbic acid or hydrochloric acid which provides a reasonably accurate TIBC even after long storage. An aqueous solution of a polycarboxylic acid has been found suitable for this purpose.

BRIEF SUMMARY OF THE INVENTION The present method consists of dissociating serum iron bytreating a serum sample with an aqueous solution of a polycarboxylic acid; removing the dissociated iron ions from the serum with an ionexchange resin; adding an excess amount of radiactive iron ions to the residual serum; measuring the initial radioactivity of the mixture; removing the iron ions not bound to transferrin from the mixture with an ion-exchange resin; measuring the radioactivity of the supernatant by means of a well-type scintillation counter; then calculating the TIBC from the initial activity and the remaining activity in the supernatant. Now, it has been confirmed that the present method can give reasonably exact TIBC values even when each reagent, particularly the polycarboxylic acid solution which has been previously prepared and stored for a long time is used.

DETAILED DESCRIPTION OF THE INVENTION Preferred polycarboxylic acids for use in practicing the present method are those containing not more than six carbon atoms, especially malonic, tartaric, malic, maleic, oxalic and citric acids. Certain of these, e.g., malic, tartaric and citric acids contain hydroxyl groups. Citric acid is particularly preferred.

A more detailed explanation of the practice of the present method is given as follows: As for the aqueous polycarboxylic acid solution (for example, citric acid, malonic acid, tartaric acid, malic acid, maleic acid or oxalic acid) which functions to reversibly dissociate the iron ions bound to serum protein, it is necessary to use sufficient acid to adjust the pH of the serum to around 4.5 so as not to impair the reversible iron-binding ability of the serum protein. For instance, about 1 ml. each of 0.6% aqueous tartaric acid solution, 0.6% aqueous citric acid solution, 0.6% aqueous malic acid solution, 0.6% aqueous malonic acid solution, 0.7% aqueous maleic acid or 0.4% aqueous oxalic acid solution is adequate for 1 ml. of serum sample. (Percentage means grams of the acid in ml. of water). In practice, it is more convenient to previously prepare and store the polycarboxylic acid solution or the same solution combined with one or more of the following ion-exchange resins to take up dissociated iron ions from the transferrin. For this purpose, use may be made of typical ionexchange resins (for example, Amberlite CG-l 20 Type 3 (cation exchanger), Amberlite CG-400 Type I (anion exchanger), Amberlite IRP67 (anion exchanger) or mixtures thereof) in powder, granule or strip form. For the solution containing radioactive iron ions, any type of radioactive iron salts can be used which can provide iron ions in a small excess of the TIBC of the serum sample (normal maximum value is about 5p.g./ml.). Usually, ferric ('Fe) ammonium citrate (in a concentration of l ugFe/ml.) is preferably used. For convenience, this radioactive iron solution may be previously combined and stored with sodium bicarbonate which neutralizes the acidic serum in order to bind the radioactive iron ions to the serum protein.

The following examples illustrate the improved results obtained with the present invention. The percent age of solution is expressed as grams of the solute in I ml. of the solvent unless otherwise defined.

EXAMPLE I The following reagents were prepared:

Solution A Portions of 0.6% aqueous citric acid solution, 0.6% aqueous tartaric acid solution, 0.6% aqueous malic acid solution, 0.6% aqueous malonic acid solution, 0.7% aqueous maleic acid solution and 0.4% aqueous oxalic acid solution (1 ml. each) were separately combined with 0.15 g. of an ion exchange resin (Amberlite CG-400 Type 1 and stored in a vial.

Solution B Separate vials were prepared, each containing 3 ml. of radioactive ferric (*Fe) ammonium citrate solution (1 g.Fe/ml.) wherein the pH was adjusted to 8.5 with sodium bicarbonate.

Solution C l.8% aqueous ascorbic acid solution.

Resin strip D A sheet of the strip contains 57.5 mg. of Amberlite IRA-400 anion exchange resin.

Resin E Amberlite CG- 1 20 Type 3 cation exchange resin (0.2 8-)- One ml. of serum sample was used, the TIBC of which had been determined to be 346 p.g./l00 ml. by the bathophenanthroline method.

Determination steps were as follows: One ml. serum was added to each vial of solution A and incubated with stirring for minutes at room temperature.

The solution was centrifuged at 3000 rpm for 10 minutes, one ml. of the supernatant was collected and added to the solution B, stirred and left to stand at room temperature for minutes. During this treatment, the radioactivity of the solution was measured by a well-type scintillation counter (N cpm). Then a sheet of strip D was added to the solution which was then set on a rotator and incubated at room temperature for 90 minutes. After the incubation, the strip D was removed and radioactivity of the residual solution was measured by the scintillation counter (N cpm).

(Amount of iron ions in the solution B pgflml.)

In the Saito method as a control run, the same procedure as above was carried out except for the use of so lution C and resin E in place of solution A and resin strip D, respectively. As a result, a significant difference was observed with time lapse in TIBC values between the test runs of this invention (solution A) and the control (solution C and resin E). For instance, when the determination procedure was done 20 days after the reagent solutions A and C were prepared, the test run using C gave TIBC values 35 to 50 pg./l00 ml. higher than the test runs using A. These data indicate that the use of preparation C during an extended period of storage gives increasingly higher TIBC values while no such trend is indicated with preparation A.

The determined TIBC values with time lapse after preparing the reagent solutions A and C are listed in Table 1.

The TIBC values of two serum samples were determined according to the following procedure:

Acid (0.5 ml. of 1N HCl or 1.0 ml. of 0.6% citric acid) was added to 1.0 ml. of serum sample. To the solution was added 0.5 g. of ion-exchange resin adsorbent (Amberlite CG-400 or Amberlite CG-l20). In the case of hydrochloric acid, 0.5 ml. of water was added to the mixture. The resulting mixture was stirred for 15 minutes and centrifuged at 3000 rpm for 10 minutes. 1.0 ml. of the supernatant was collected. To the fluid was added 1.0 ml. of ferric ammonium citrate ("Fe) (2.47 pg. Fe/ml.). Then, the radioactivity of the solution was measured (N To the solution was added 1.4 ml. of 0.2N tris buffer (in the case where HCl was used) or 1.0 ml. of 2% sodium bicarbonate (in the case where citric acid was used) and water up to 4.0 ml. The resulting solutions were allowed to stand for 20 minutes. After addition of 0.5 g. of ion-exchange resin (Amberlite CG-400), the mixtures were stirred for 15 minutes and centrifuged at 3000 rpm for l0 minutes. Two milliliters of the supernatant were collected in each case and the radioactivity was measured by a well-type scintillation counter (N,). The TIBC value was calculated from the equation.

2 N -N i+ 2.47 we x 200 wherein N, indicates the background count.

The results are shown in Table 2.

Table 2 TIBC value (m g/ ml.)

lN HCl Table 2-continued TlBC value [mtg/I00 ml.)

lN HCl lN HCl Sample 0.6% citric Amber Ambernumber acid Amberlite lite of serum lite CG-400 C6400 CG-l20 tial activity of the resulting mixture, removing the unbound ferric ions, and determining the residual radioactivity, the improvement which comprises adding a polycarboxylic acid selected from the group consisting of malonic, tartaric, malic, maleic and oxalic acids to the blood serum specimen so as to release the bound iron from the transferrin therein.

2. The method of claim 1 wherein the polycarboxylic acid is malonic acid at a concentration of 0.6%.

3. The method of claim 1 wherein the polycarboxylic acid is tartaric acid at a concentration of 0.6%.

4. The method of claim 1 wherein the polycarboxylic acid is malic acid at a concentration of 0.6%.

5. The method of claim 1 wherein the polycarboxylic acid is maleic acid at a concentration of 0.7%.

6. The method of claim 1 wherein the polycarboxylic acid is oxalic acid at a concentration of 0.4%. 

1. IN THE METHOD FOR DETERMINING THE TOTAL IRON-BINDING CAPACITY OF BLOOD SERUM WHICH COMPRISES ADDING AN ACID TO A BLOOD SERUM SPECIMEN TO RELEASE THE BOUND IRON FROM THE TRANSFERRIN, REMOVING THE RELEASED IRON, RETURNING THE PH OF THE REMAINING SERUM FRACTION TO THE RANGE WHEREIN QUANTITATIVE BINDING OF FERRIC IONS TO TRANSFERRIN OCCURS, ADDING AN EXCESS OF FERRIC IONS INCLUDING SOME RADIOACTIVE FERRIC IONS, MEASURING THE INITIAL ACTIVITY OF THE RESULTING MIXTURE, REMOVING THE UNBOUND FERRIC IONS, AND DETERMINING THE RESIDUAL RADIOACTIVITY, THE IMPROVEMENT WHICH COMPRISES ADDING A POPOLYCARBOXYLIC ACID SELECTED FROM THE GROUP CONSISTING OF MALONIC, TARTARIC, MALIC, MALEIC AND OXALIC ACIDS TO THE BLOOD SERUM SPECIMEN SO AS TO RELEASE THE BOUND IRON FROM THE TRANSFERRIN THEREIN.
 2. The method of claim 1 wherein the polycarboxylic acid is malonic acid at a concentration of 0.6%.
 3. The method of claim 1 wherein the polycarboxylic acid is tartaric acid at a concentration of 0.6%.
 4. The method of claim 1 wherein the polycarboxylic acid is malic acid at a concentration of 0.6%.
 5. The method of claim 1 wherein the polycarboxylic acid is maleic acid at a concentration of 0.7%.
 6. The method of claim 1 wherein the polycarboxylic acid is oxalic acid at a concentration of 0.4%. 